JP7426200B2 - Method for producing glutamic acid-5-semialdehyde using recombinant microorganisms - Google Patents

Description

NPMD FERM BP-11515 NPMD NITE P-02919 NPMD FERM BP-11512

The present invention relates to a recombinant microorganism that has been artificially genetically engineered to produce industrially useful compounds, and the use of the recombinant microorganism to produce glutamic acid-5-semialdehyde, pyrroline-5-carboxylic acid, and L. - Concerning a method for producing one or more of pyroglutamic acids.

Glutamic acid-5-semialdehyde (CAS No. 2886-91-1) is used, for example, as a raw material for organic synthesis, a polyamide raw material intermediate, a polyurethane raw material intermediate, an isocyanate raw material intermediate, a PET raw material intermediate, and a solvent raw material intermediate. It is an important compound (Patent Documents 1 to 4). In vivo, glutamic acid-5-semialdehyde is produced starting from L-glutamic acid and via L-glutamic acid-5-phosphate as an intermediate metabolite (see FIG. 1). Enzymatically, it is synthesized by treatment with ornithine aminotransferase using L-ornithine and 2-oxoglutaric acid as substrates (Non-Patent Document 1). Furthermore, an example of obtaining a natural microorganism that produces glutamic acid-5-semialdehyde has been reported, but the biosynthetic mechanism thereof has not been specified (Patent Document 5).

Pyrroline-5-carboxylic acid (CAS No. 2906-39-0) is a compound produced by intramolecular condensation of glutamic acid-5-semialdehyde. By being converted to glutamic acid-5-semialdehyde, it can be used for the purposes mentioned above for glutamic acid-5-semialdehyde, and the present compound itself is also a useful compound as a herbicide raw material and a pharmaceutical intermediate raw material ( Patent Document 6, Non-Patent Document 2). In vivo, it is produced by spontaneous cyclization of glutamate-5-semialdehyde and is enzymatically reduced to proline, an essential amino acid. In organic synthesis, for example, it is synthesized by heating (S)-5-Hydroxy-2-aminovaleric acid to 80°C in the presence of chromium (IV) oxide under acidic conditions, but chromium (IV) oxide It is designated as a Class 1 designated chemical under the Chemical Substances Control Law, and its use is subject to severe restrictions (Non-patent Document 3). Although the two compounds mentioned above are industrially important compounds, useful production methods have not yet been established.

L-pyroglutamic acid (CAS No. 98-79-3) is a compound produced by intramolecular condensation of L-glutamic acid. Since L-pyroglutamic acid is harmless to living organisms, it has been put into practical use as a moisturizing agent for external skin medicines and cosmetics for the purpose of moisturizing (Patent Documents 7 to 9). In organic synthesis, L-pyroglutamic acid is synthesized by heating and dehydrating L-glutamic acid at 175° C. under acidic conditions. However, this manufacturing method is undesirable because the heating temperature is extremely high temperature and pressure of 175° C., and it is performed under acidic conditions (Non-Patent Document 4). As another method for synthesizing L-pyroglutamic acid, a method for enzymatically producing it from L-glutamic acid has been reported (Patent Document 10).

Patent No. 1210190 Special Publication No. 2015-519083 Patent No. 5773990 Patent No. 5912529 Japanese Unexamined Patent Publication No. 53-136586 Patent No. 4418540 Special Publication No. 3-29764 Patent No. 4522580 Japanese Patent Application Publication No. 61-60620 Patent No. 2698056

Stranska et. Al. Biochemical characterization of pea ornithine-δ-aminotransferase: Substrate specificity and inhibition by di- and polyami nes, Biochimie, Holland, Elsevier Masson SAS, 2010, 92, 940-948. Hamed et. al. Stereoselective preparation of lipidated carboxymethylproline/pipecolic acid derivatives via coupling of engineered crotona ses with an alkylmalonyl-CoA synthetase, Organic & Biomolecular Chemistry, UK, The Royal Society of Chemistry, 21 December 201 3, 11, 47, 8191-8196 Luesch et. al. Biosynthesis of 4-Methylproline in Cyanobacteria: Cloning ofnosE and nosF Genes and Biochemical Characterization of the E Coded Dehydrogenase and Reductase Activities, Journal of Organic Chemistry, USA, American Chemical Society, 2003, 68, 83-91 Beecham et. Al. L-Pyrrolidonecarboxylic Acid. Journal of the American Chemical Society, ACS Publication, September 1954, 76, 18, 4613-4614.

The problem of the present invention is to use genetically engineered microorganisms to produce one of glutamic acid-5-semialdehyde, pyrroline-5-carboxylic acid and L-pyroglutamic acid from sugar, which is a cheap and stable raw material. The purpose is to provide a method for producing the above.

In order to solve the above-mentioned problems, the present inventors have conducted intensive research and found that by manipulating genes related to the L-proline biosynthetic pathway of microorganisms, the present inventors can convert sugar into glutamate-5-semialdehyde, pyrroline-5-semialdehyde, and pyrroline- It has been found that recombinant microorganisms can be obtained that are capable of producing one or more of 5-carboxylic acid and L-pyroglutamic acid.

That is, the present invention provides:
[1] Modification such that the expression of the gene encoding pyrroline-5-carboxylic acid dehydrogenase and the gene encoding pyrroline-5-carboxylic acid reductase is suppressed, or the activity of these enzymes is suppressed recombinant microorganisms;
[2] The recombinant microorganism according to [1], which has been further modified to enhance the activity of glutamate-5-phosphate synthase;
[3] The recombinant microorganism according to [2], wherein the modification releases feedback inhibition of glutamate-5-phosphate synthase;
[4] The gene encoding the pyrroline-5-carboxylic acid dehydrogenase,
・DNA consisting of the base sequence shown in SEQ ID NO: 27,
・DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 27, and encodes a protein that has pyrroline-5-carboxylic acid dehydrogenase activity,
- A DNA consisting of a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 27 and encoding a protein having pyrroline-5-carboxylic acid dehydrogenase activity,
- DNA encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted, and/or added to the amino acid sequence of the protein encoded by the base sequence shown in SEQ ID NO: 27. DNA encoding a protein having pyrroline-5-carboxylic acid dehydrogenase activity, or DNA consisting of a degenerate isomer of the base sequence shown in SEQ ID NO: 27.
and
and/or
The gene encoding the pyrroline-5-carboxylic acid reductase is
・DNA consisting of the base sequence shown in SEQ ID NO: 28,
- DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 28, and encodes a protein that has pyrroline-5-carboxylic acid reductase activity ,
- A DNA consisting of a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 28 and encoding a protein having pyrroline-5-carboxylic acid reductase activity,
- DNA that encodes a protein consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted, and/or added to the amino acid sequence of the protein encoded by the base sequence shown in SEQ ID NO: 28. DNA encoding a protein having pyrroline-5-carboxylic acid reductase activity, or DNA consisting of a degenerate isomer of the base sequence shown in SEQ ID NO: 28.
The recombinant microorganism according to any one of [1] to [3], which is;
[5] The pyrroline-5-carboxylic acid dehydrogenase is selected from the enzymes represented by EC 1.2.1.88, and/or the pyrroline-5-carboxylic acid reductase is selected from the enzymes represented by EC 1.5.1. The recombinant microorganism according to any one of [1] to [4], which is selected from enzymes represented by 2.

[6] The pyrroline-5-carboxylic acid dehydrogenase has an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and the pyrroline-5-carboxylic acid dehydrogenase It is a protein with activity,
and/or
The pyrroline-5-carboxylic acid reductase is a protein having an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 24 and having pyrroline-5-carboxylic acid reductase activity. The recombinant microorganism according to any one of [1] to [5];
[7] The recombinant microorganism according to any one of [1] to [6], wherein the microorganism belongs to the genus Escherichia;
[8] The recombinant microorganism according to any one of [1] to [7], wherein the microorganism is Escherichia coli;
[9] The glutamic acid-5-phosphate synthase,
・DNA consisting of a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 29 and encoding a protein having glutamic acid-5-phosphate synthase activity, or
- DNA that encodes a protein consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted, and/or added to the amino acid sequence of the protein encoded by the base sequence shown in SEQ ID NO: 29. DNA encoding a protein having glutamate-5-phosphate synthase activity
The recombinant microorganism according to any one of [2] to [8], which is encoded by;
[10] Producing a protein having an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 25 and having glutamate-5-phosphate synthase activity [2] to [ The recombinant microorganism according to any one of [9];
[11] For the amino acid sequence shown in SEQ ID NO: 25,
Substitution of the 107th aspartic acid residue with an asparagine residue,
has an amino acid sequence that has 80% or more sequence identity to an amino acid sequence containing a substitution of an alanine residue at position 117 with a valine residue and a substitution of a glutamic acid residue at position 143 with a lysine residue, and the recombinant microorganism according to any one of [2] to [10], which produces a protein having glutamate-5-phosphate synthase activity;
[12] For the amino acid sequence shown in SEQ ID NO: 25,
Substitution of the 107th aspartic acid residue with an asparagine residue,
has one or more mutations selected from the group consisting of substitution of the 117th alanine residue with a valine residue, and substitution of the 143rd glutamic acid residue with a lysine residue, and glutamic acid-5-phosphorus The recombinant microorganism according to any one of [2] to [11], which produces a protein having acid synthase activity;
[13] According to any one of [2] to [12], the gene encoding glutamate-5-phosphate synthase is included as an operon with the gene encoding glutamate-5-semialdehyde reductase. The recombinant microorganisms described;
[14] A method for producing glutamic acid-5-semialdehyde, which comprises culturing the recombinant microorganism according to any one of [1] to [13];
[15] A method for producing pyrroline-5-carboxylic acid, which comprises culturing the recombinant microorganism according to any one of [1] to [13];
[16] A method for producing L-pyroglutamic acid, which comprises culturing the recombinant microorganism according to any one of [1] to [13].

According to the present invention, one or more of glutamic acid-5-semialdehyde, pyrroline-5-carboxylic acid, and L-pyroglutamic acid can be produced from sugar, which is an inexpensive and stable raw material.

The proline biosynthesis pathway within microbial cells is shown. The amino acid sequence (residues 1 to 760) of the PutA enzyme derived from E. coli strain W3110 is shown. The amino acid sequence (residues 761 to 1320) of the PutA enzyme derived from E. coli strain W3110 is shown. The amino acid sequence of ProC enzyme derived from E. coli strain W3110 is shown. The amino acid sequence of ProB enzyme derived from E. coli strain W3110 is shown. The amino acid sequence of ProA enzyme derived from E. coli strain W3110 is shown. 3 shows a LCMS analysis TIC chart of the culture supernatant in Example 3-1. 3 shows an LCMS analysis TIC chart of the culture supernatant in Example 3-2.

EMBODIMENT OF THE INVENTION Hereinafter, the form for implementing this invention is demonstrated concretely. Note that the present invention is not limited to the following embodiments, and can be implemented with various modifications within the scope of the gist.

In the recombinant microorganism according to the present invention, the expression of the gene encoding pyrroline-5-carboxylic acid dehydrogenase and the gene encoding pyrroline-5-carboxylate reductase is suppressed, or the activity of these enzymes is suppressed. Modifications have been made to suppress this. That is, in the recombinant microorganism of the present invention, the expression of the gene encoding pyrroline-5-carboxylic acid dehydrogenase and the gene encoding pyrroline-5-carboxylic acid reductase is suppressed in the host microorganism, or Alternatively, the enzymes are modified so that the activity of these enzymes is suppressed. Here, the modification includes base substitution, deletion, insertion, and/or addition.

<1> Pyrroline-5-carboxylic acid dehydrogenase "Pyrroline-5-carboxylic acid dehydrogenase" has one or more amino acids deleted, substituted, inserted, and/or added in the amino acid sequence of the enzyme. Also included are proteins containing the amino acid sequence of the enzyme, which are functionally equivalent to the enzyme. Here, a "functionally equivalent protein" is a protein that has an activity similar to that of the enzyme or protein. For example, a "functionally equivalent protein" includes a protein that has 80%, 85%, 90%, 95%, 97%, 98% or 99% or more sequence identity with the amino acid sequence of the enzyme. Specifically, "pyrroline-5-carboxylic acid dehydrogenase" is 80%, 85%, 90%, 95%, 97%, 98% or 99% or more of the amino acid sequence shown in the SEQ ID NO. specified below. It includes proteins having an amino acid sequence having sequence identity with , and having pyrroline-5-carboxylic acid dehydrogenase activity. In a preferred embodiment of the invention, the pyrroline-5-carboxylic acid dehydrogenase is selected from enzymes with EC 1.2.1.88.

"The gene encoding pyrroline-5-carboxylic acid dehydrogenase" is
・DNA consisting of the base sequence shown in the sequence number specified below,
- Hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence shown in the sequence number specified below, and encodes a protein that has pyrroline-5-carboxylic acid dehydrogenase activity. DNA,
・Pyrroline-5-carboxylic acid that consists of a base sequence that has 85%, 90%, 95%, 97%, 98% or 99% or more sequence identity with the base sequence shown in the sequence number specified below, and DNA encoding a protein with dehydrogenase activity,
- One or more (for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, A DNA encoding a protein consisting of an amino acid sequence in which 1 to 3 amino acids, more preferably 1 or 2 amino acids, are deleted, substituted, inserted and/or added, wherein DNA encoding a protein with acid dehydrogenase activity, and DNA consisting of a degenerate isomer of the base sequence shown in the sequence number specified below.
includes.

In the present invention, "stringent conditions" are, for example , conditions such as " 0.1x SSC, 0.1% SDS, 68°C", which are washing conditions for Southern hybridization.

<2> Pyrroline-5-carboxylate reductase "Pyrroline-5-carboxylate reductase" has one or more amino acids deleted, substituted, inserted, and/or added in the amino acid sequence of the enzyme. Also included are proteins containing an amino acid sequence that are functionally equivalent to the enzyme. Here, the "functionally equivalent protein" is the same as that described for "pyrroline-5-carboxylic acid dehydrogenase." Specifically, "pyrroline-5-carboxylic acid reductase" has 80%, 85%, 90%, 95%, 97%, 98% or 99% or more of the amino acid sequence shown in the sequence number specified below. It includes proteins that have amino acid sequences with sequence identity and have pyrroline-5-carboxylic acid reductase activity. In one preferred embodiment of the invention, the pyrroline-5-carboxylic acid reductase is selected from enzymes with EC 1.5.1.2.

"The gene encoding pyrroline-5-carboxylic acid reductase" is
・DNA consisting of the base sequence shown in the sequence number specified below,
・DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence shown in the sequence number specified below, and that encodes a protein that has pyrroline-5-carboxylic acid reductase activity.
・Pyrroline-5-carboxylic acid that consists of a base sequence that has 85%, 90%, 95%, 97%, 98% or 99% or more sequence identity with the base sequence shown in the sequence number specified below, and DNA encoding a protein with reductase activity,
- One or more (for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, A DNA encoding a protein consisting of an amino acid sequence in which 1 to 3 amino acids, more preferably 1 or 2 amino acids, are deleted, substituted, inserted and/or added, wherein DNA encoding a protein with acid reductase activity, and DNA consisting of a degenerate isomer of the base sequence shown in the sequence number specified below.
includes.

Essentially any host microorganism that can be used in the present invention can be used as long as it has the L-proline biosynthetic pathway shown in FIG. Examples of such microorganisms include bacteria such as Escherichia, Pseudomonas, Bacillus, Corynebacterium, Brevibacterium, Micrococcus, Flavobacterium, Erwinia, Vibrio, Rhodotorula, Saccharomyces, and Candida. Among these, Escherichia genus is preferred, and Escherichia coli W3110 strain is more preferred.

In the present invention, "suppression of activity" with respect to enzymes is synonymous with "suppression of function," "reduction of function," and "reduction of activity," and are used interchangeably. "Suppression of enzyme activity" means that the activity per bacterium is lower than the activity of the unmodified strain. For example, the number of protein molecules per bacterium is reduced, or the specific activity per protein is reduced.

One way to obtain microorganisms with suppressed enzyme activity is to induce random mutations in their genomic DNA by irradiating them with ultraviolet light or treating them with mutagenic chemicals, and then screening for strains that have the desired mutations. . Alternatively, if the gene sequence is known in advance, this can also be achieved by removing part or all of the genes in the bacterial chromosome using techniques such as homologous recombination and genome editing. Alternatively, the promoter sequence involved in the expression of the target gene may be changed to one with lower expression efficiency. In any case, the method is not limited to the exemplified one.

Hereinafter, the present invention will be described in further detail with reference to the drawings.

Pyrroline-5-carboxylic acid dehydrogenase (for example, expressed by EC 1.2.1.88), as shown in FIG. Glutamic acid is produced by oxidizing the aldehyde group of glutamic acid semialdehyde, which is spontaneously produced by the addition of a water molecule to pyrroline-5-carboxylic acid, using NAD as a coenzyme. Information regarding the amino acid sequence of pyrroline-5-carboxylic acid dehydrogenase and the base sequence of the gene encoding the enzyme can be obtained from databases known in the art. A representative gene includes putA of Escherichia coli. The amino acid sequence of the E. coli PutA enzyme is shown in Figures 2 and 3 (SEQ ID NO: 23). Furthermore, the base sequence information of E. coli putA is also available from, for example, GenBank, and one example is Gene ID: 945600 (SEQ ID NO: 27).

Pyrroline-5-carboxylic acid reductase (e.g., represented by EC 1.5.1.2) catalyzes the reaction that reduces pyrroline-5-carboxylic acid to produce proline, as shown in Figure 1. . Information regarding the amino acid sequence of pyrroline-5-carboxylic acid reductase and the base sequence of the gene encoding the enzyme can be obtained from databases known in the art. A typical gene sequence is proC of Escherichia coli. The amino acid sequence of E. coli ProC enzyme is shown in Figure 4 (SEQ ID NO: 24). Furthermore, the base sequence information of E. coli proC is also available from, for example, GenBank, and one example is Gene ID: 945034 (SEQ ID NO: 28).

By suppressing the expression of the genes encoding the above two enzymes or by modifying them to suppress the activity of these enzymes, proline of pyrroline-5-carboxylic acid in the proline biosynthesis pathway It is possible to suppress the conversion to L-glutamic acid and improve the yield of the target compound of the present invention.

In a preferred embodiment of the present invention, in addition to suppressing the expression or enzyme activity of the aforementioned genes, the activity of glutamate-5-phosphate synthase is enhanced in order to promote glutamate-5-semialdehyde biosynthesis in the host microorganism. Changes have been made to ensure that. The activity of the enzyme can be enhanced, for example, by introducing a gene encoding glutamate-5-phosphate synthase in addition to the gene encoding glutamate-5-phosphate synthase that the host microorganism originally has. or by introducing a mutation into the glutamate-5-phosphate synthase gene originally possessed by the host microorganism. When introducing a gene encoding glutamate-5-phosphate synthase in addition to the gene encoding glutamate-5-phosphate synthase that the host microorganism originally possesses, mutated glutamate-5-phosphate A gene encoding an acid synthase (hereinafter also referred to as "glutamic acid-5-phosphate synthase mutant gene") is introduced into a host microorganism. When introducing a glutamate-5-phosphate synthase mutant gene into a host microorganism, the enzyme may be introduced alone, and the glutamate-5-semialdehyde reductase described below (for example, EC 1.2.1 It may also be introduced as an operon with a gene encoding (expressed as .41).

Glutamic acid-5-phosphate synthase catalyzes a reaction that phosphorylates the 5-position carboxylic acid of glutamic acid to produce glutamic acid-5-phosphate. Here, "glutamate-5-phosphate synthase" refers to a protein containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and/or added to the amino acid sequence of the enzyme. , proteins that are functionally equivalent to the enzyme are also included. The "functionally equivalent protein" is the same as that described for "pyrroline-5-carboxylic acid dehydrogenase" and "pyrroline-5-carboxylic acid reductase." Specifically, "glutamate-5-phosphate synthase" has 80%, 85%, 90%, 95%, 97%, 98% or 99% or more of the amino acid sequence shown in the SEQ ID NO. specified below. It includes proteins that have amino acid sequences with sequence identity and have glutamate-5-phosphate synthase activity. In one preferred embodiment of the invention, the glutamate-5-phosphate synthase is selected from enzymes with EC 2.7.2.11.

"The gene encoding glutamate-5-phosphate synthase" is
・DNA consisting of the base sequence shown in the sequence number specified below,
・DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence shown in the sequence number specified below, and that encodes a protein that has glutamate-5-phosphate synthase activity.
・Consists of a base sequence having 85%, 90%, 95%, 97%, 98% or 99% or more sequence identity with the base sequence shown in the sequence number specified below, and glutamic acid-5-phosphate DNA encoding a protein with synthetic enzyme activity,
- One or more (for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, DNA encoding a protein consisting of an amino acid sequence in which glutamic acid-5-phosphate DNA encoding a protein with acid synthase activity, and DNA consisting of a degenerate isomer of the base sequence shown in the sequence number specified below.
includes.

A representative gene includes proB of Escherichia coli. The amino acid sequence of E. coli ProB enzyme is shown in Figure 5 (SEQ ID NO: 25). Furthermore, the base sequence information of E. coli proB is also available from, for example, GenBank, and one example is Gene ID: 946425 (SEQ ID NO: 29).

Glutamic acid-5-semialdehyde reductase (for example, expressed by EC 1.2.1.41) converts the aldehyde group while removing the phosphoric acid modified from the carboxyl group at the 5-position of L-glutamic acid-5-phosphate. catalyze reactions that produce "Glutamic acid-5-semialdehyde reductase" refers to a protein containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and/or added to the amino acid sequence of the enzyme; Also includes proteins that are functionally equivalent. Specifically, "glutamate-5-semialdehyde reductase" has 80%, 85%, 90%, 95%, 97%, 98% or 99% or more of the amino acid sequence shown in the SEQ ID NO. specified below. It includes proteins that have amino acid sequences with sequence identity and have glutamate-5-semialdehyde reductase activity. In addition, "the gene encoding glutamate-5-semialdehyde reductase" is
・DNA consisting of the base sequence shown in the sequence number specified below,
・DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence shown in the sequence number specified below, and that encodes a protein that has glutamate-5-phosphate synthase activity.
・Glutamic acid-5-semialdehyde that consists of a base sequence that has 85%, 90%, 95%, 97%, 98% or 99% or more sequence identity with the base sequence shown in the sequence number specified below DNA encoding a protein with reductase activity,
- One or more (for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, A DNA encoding a protein consisting of an amino acid sequence in which glutamic acid-5-semi DNA encoding a protein having aldehyde reductase activity, and DNA consisting of a degenerate isomer of the base sequence shown in the sequence number specified below.
includes. A representative gene includes proA of Escherichia coli. The amino acid sequence of E. coli ProA enzyme is shown in Figure 6 (SEQ ID NO: 26). Furthermore, the base sequence information of E. coli proA is also available from, for example, GenBank, and one example is Gene ID: 946680 (SEQ ID NO: 30).

It is generally recognized that wild-type glutamate-5-phosphate synthase is feedback inhibited by L-proline. Therefore, it is preferable to have a mutation that releases feedback inhibition. An example of a microorganism that highly expresses a mutant glutamate-5-phosphate synthase whose feedback inhibition has been released through genetic recombination has been reported (Patent No. 4637183, Jiang et. al., Nature COMMUNICATIONS, SPRINGER NATURE, 2017, Dandekar et. al., JOURNAL OF BACTERIOLOGY, American society for microbiology, 1988, 170, 12, 5943-594 5.).

Hereinafter, proB derived from Escherichia coli W3110 will be explained as an example of glutamate-5-phosphate synthase, but the gene used in the present invention is not limited to this. Mutant ProBs that are not subject to feedback inhibition by L-proline include those in which the 107th aspartic acid residue in the amino acid sequence is replaced with an asparagine residue, and the 117th alanine residue in the amino acid sequence is replaced with a valine residue. Examples include those in which the glutamic acid residue at position 143 is replaced with a lysine residue. However, the types of amino acids used for substitution are not limited to these. Moreover, the number of mutation points may be one, or a combination of multiple points may be used. Mutant glutamate-5-phosphate synthase that is not subject to feedback inhibition by L-proline has an amino acid sequence having the above amino acid substitution and 80%, 85%, 90%, 95%, 97%, 98% or 99%. Included are proteins that have amino acid sequences with % or more sequence identity and have glutamate-5-phosphate synthase activity.

In the present invention, more stable and high-level enzyme activity can be obtained by introducing the above-mentioned glutamate-5-phosphate synthase mutant gene into host microorganism cells as an "expression cassette." As used herein, "expression cassette" refers to a nucleotide containing a nucleic acid sequence that regulates transcription and translation operably linked to a nucleic acid to be expressed or a gene to be expressed. Typically, the expression cassettes of the invention will include in operably linked a promoter sequence 5' upstream from the coding sequence, a terminator sequence 3' downstream, and optionally further conventional regulatory elements; In some cases, a nucleic acid to be expressed or a gene to be expressed is introduced into a host microorganism.

The above promoters include the lac system, trp system, tac or trc system in E. coli, the main operator and promoter region of λ phage, the control region of fd coat protein, glycolytic enzymes (e.g. 3-phosphoglycerate kinase, glycerol Promoters for aldehyde-3-phosphate dehydrogenase), glutamic acid decarboxylase A, and serine hydroxymethyltransferase are available. As the terminator, rrnBT1T2 terminator, lac terminator, etc. can be used. Besides promoter and terminator sequences, examples of other regulatory elements may be mentioned such as selection markers, amplification signals, origins of replication, etc. Suitable regulatory sequences are described, for example, in "Gene Expression Technology: Methods in Enzymology 185", Academic Press (1990).

The expression cassette described above is incorporated into a vector consisting of, for example, a plasmid, a phage, a transposon, an IS element, a phasmid, a cosmid, or a linear or circular DNA, and is inserted into a host microorganism. Plasmids and phages are preferred in the present invention. These vectors may be autonomously replicated in the host microorganism, or may be inserted into the chromosome and replicated. Suitable plasmids include, for example, pLG338, pACYC184, pBR322, pUC18, pUC19, pHSG298, pHSG398, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG20 in E. coli. 0, pUR290, pIN-III113 -B1, λgt11 or pBdCI. Plasmids and the like that can be used in addition to these are described in "Cloning Vectors", Elsevier, 1985. Introduction of the expression cassette into a vector is possible by conventional methods including fragment amplification by PCR, excision with appropriate restriction enzymes, cloning, and various ligations.

After the vector having the expression cassette of the present invention is constructed as described above, methods that can be applied to introduce the vector into a host microorganism include coprecipitation, protoplast fusion, electroporation, and retrovirus transfection. Conventional cloning and transfection methods are used, such as. Examples of these are "Current Protocols in Molecular Biology", F. Ausubel et al., Publ. Wiley Interscience, New York, 1997, or Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press. , Cold Spring Harbor, NY, 1989.

Another embodiment of the invention relates to a method of producing one or more of glutamic acid-5-semialdehyde, pyrroline-5-carboxylic acid and L-pyroglutamic acid using the aforementioned recombinant microorganism. For example, in one embodiment, glutamic acid-5-semialdehyde, pyrroline-5-carboxylic acid, and L-pyroglutamic acid are produced by culturing the aforementioned recombinant microorganism. In another embodiment, glutamic acid-5-semialdehyde and pyrroline-5-carboxylic acid are produced by culturing the aforementioned recombinant microorganism. In yet another embodiment, L-pyroglutamic acid is produced by culturing the aforementioned recombinant microorganism.

Suitable medium composition, culture conditions, and culture time for the recombinant microorganism according to the present invention can be appropriately selected by those skilled in the art.

The medium may be a natural, semi-synthetic, or synthetic medium containing one or more carbon sources, nitrogen sources, inorganic salts, vitamins, and optionally trace components such as trace elements or vitamins. The medium used must adequately meet the nutritional requirements of the transformants to be cultured.

Carbon sources include D-glucose, sucrose, lactose, fructose, maltose, oligosaccharides, polysaccharides, starch, cellulose, rice bran, waste molasses, oils and fats (such as soybean oil, sunflower oil, peanut oil, coconut oil, etc.), and fatty acids. (eg, palmitic acid, linoleic acid, linolenic acid, etc.), alcohols (eg, glycerol, ethanol, etc.), and organic acids (eg, acetic acid, lactic acid, succinic acid, etc.). It may also be a biomass containing D-glucose. Examples of suitable biomass include corn decomposition liquid and cellulose decomposition liquid. These carbon sources can be used individually or as a mixture. Glucose is particularly preferred as the carbon source.

Nitrogen sources include nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour, and urea) or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, phosphoric acid, etc.). ammonium, ammonium carbonate, sodium nitrate, ammonium nitrate, etc.). These nitrogen sources can be used individually or in mixtures.

The medium may also contain a corresponding antibiotic if the transformant expresses a useful additional trait, for example if it has a marker for resistance to the antibiotic. This reduces the risk of contamination by bacteria during fermentation. Antibiotics include, but are not limited to, ampicillin, kanamycin, chloramphenicol, tetracycline, erythromycin, streptomycin, spectinomycin, and the like.

If the host microorganism is unable to assimilate carbon sources such as cellulose and polysaccharides, known genetic engineering techniques such as introducing foreign genes into the host microorganism can be used to produce glutamic acid using these carbon sources. It can be adapted to produce 5-semialdehyde and proline-5-carboxylic acid, and L-pyroglutamic acid. Examples of foreign genes include cellulase genes and amylase genes.

Cultivation may be a batch method or a continuous method. Furthermore, in any case, the above-mentioned carbon source, etc. may be supplemented at an appropriate point in culture. Furthermore, culturing should be continued while maintaining suitable temperature, oxygen concentration, pH, etc. Suitable culture temperatures for transformants derived from common microbial host cells are usually in the range of 15°C to 45°C, preferably 25°C to 37°C. If the host microorganism is aerobic, shaking (e.g., flask culture) and agitation/aeration (e.g., jar fermenter culture) may be necessary to ensure appropriate oxygen concentrations during fermentation. Those culture conditions can be easily set by those skilled in the art.

Given the above description, one skilled in the art will be able to fully practice the present invention. Examples are given below for the purpose of further explanation and the invention is therefore not limited to these examples. In this specification, unless otherwise specified, nucleotide sequences are written in the 5' to 3' direction.

All PCRs shown in this example were performed using PrimeSTAR Max DNA Polymerase (Takara Bio). E. coli was transformed using the calcium chloride method (Yodosha Genetic Engineering Experiment Notes Vol. 1, written by Takaaki Tamura).

In constructing the plasmid, LB medium was used with the addition of necessary antibiotics. Table 1 shows the primer sequences used in each example.

The composition of the medium used in each example is as follows.

(LB medium)
20g/L LB medium, Lennox (manufactured by Difco).

[Steam sterilization was performed at 120°C for 20 minutes. If necessary, ampicillin at a final concentration of 100 mg/L, kanamycin sulfate at a final concentration of 50 mg/L, chloramphenicol at a final concentration of 30 mg/mL, and 1.5% Bacto agar (manufactured by Difco) were added.

(M9 medium)
6g/L KH ₂ PO ₄ , 12g/L
_Na2HPO4 , _1g /L
NaCl, 2g/L
_NH4Cl ,
2mM _{MgSO4.7H2O ,}
0.2mM _CaCl2 ,
Glucose 20g/L,
1mL trace metal solution _{( 500mg} /L FeSO4.7H2O, 400mg/ _{L ZnSO4.7H2O} , 20mg/L _MnCl2.7H2O , 50mg/L _CoCl2.6H2O , _10mg /L NiCl _{2.6H 2} O, 15 mg/L H ₃ BO ₃ , 250 mg/L EDTA),
100mg/L thiamin,
1g/L Yeast Extract
[If necessary, ampicillin at a final concentration of 100 mg/L, or kanamycin sulfate at a final concentration of 50 mg/L, or chloramphenicol at a final concentration of 30 mg/mL, and L-arabinose at a final concentration of 250 mg/mL were also added. KH ₂ PO ₄ , Na ₂ HPO ₄ , NaCl, and NH ₄ Cl were mixed and steam sterilized at 120° C. for 20 minutes. Trace metal solution and Yeast Extract were individually steam sterilized at 120° C. for 20 minutes and then mixed. MgSO ₄ , CaCl ₂ , glucose, and thiamine were individually filter-sterilized and then mixed. ]
[Example 1] Construction of a plasmid (1-1) Construction of a constitutive expression plasmid A gene constitutive expression vector used in E. coli was constructed as follows. Plasmid pNFP-A51 (as FERM P-22182) was submitted to the National Institute of Technology and Evaluation (Independent Administrative Agency) Patent Organism Depositary Center (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on October 25, 2011. The terminator sequence and promoter sequence were inserted into the multi-cloning site of International Deposit Number: FERM BP-11515) to construct pSK000. The promoter sequence (SEQ ID NO: 21) and terminator sequence (SEQ ID NO: 22) are shown in Table 2.

E. coli strain W3110 (NBRC12713) was cultured with shaking in LB medium (2 ml) at 37°C. After completion of the culture, bacterial cells were collected from the culture solution, and genomic DNA was extracted using a Nucleo Spin Tissue. Using the extracted genomic DNA as a template, the glutamate-5-phosphate synthase gene and the glutamate-5-phosphate synthase gene/glutamate-5-semialdehyde reductase gene operon were PCR amplified. The primer set described in SEQ ID NOs: 1 and 2 shown in Table 1 was used for proB gene amplification. The primer set described in SEQ ID NOs: 1 and 3 shown in Table 1 was used to amplify the proB/proA operon gene sequence. The reaction conditions for proB gene amplification were 98°C (10sec), 55°C (5sec), 72°C (10sec), and 30 cycles. The reaction conditions for proB/proA operon gene amplification were 98°C (10sec), 55°C (5sec), 72°C (15sec), and 30 cycles. The obtained proB coding region and proB/proA operon coding region were phosphorylated using Mighty Cloning Reagent Set (manufactured by Takara), ligated downstream of the promoter of pSK000 plasmid, and transformed into E. coli JM109 strain. Plasmids were extracted from the colonies that appeared and named pSK001 and pSK004, respectively.

Mutations were introduced into the glutamate-5-phosphorus synthase gene sequence using an inverse PCR method. First, mutations were introduced for the purpose of replacing the 107th aspartic acid residue with an asparagine residue and the 117th alanine residue with a valine residue. That is, inverse PCR was performed using the pSK001 plasmid or pSK004 plasmid as a template and the primer set shown in SEQ ID NOs: 4 and 5 shown in Table 1. The reaction conditions for proB gene amplification were 98°C (10sec), 55°C (5sec), 72°C (10sec), and 30 cycles. The reaction conditions for proB/proA operon gene amplification were 98°C (10sec), 55°C (5sec), 72°C (15sec), and 30 cycles. The obtained PCR fragment was phosphorylated using Mighty Cloning Reagent Set (manufactured by Takara), self-ligated, and transformed into E. coli JM109 strain. Plasmids were extracted from the colonies that appeared and named pSK002 and pSK005, respectively. Hereinafter, the proB gene into which the two mutations described above have been introduced will be referred to as proB2m .

Using the pSK005 plasmid as a template, mutation introduction was performed to replace the 143rd glutamic acid residue with a lysine residue. First, inverse PCR was performed using the primer set shown in SEQ ID NOs: 6 and 7 shown in Table 1. The reaction conditions were 98°C (10sec), 55°C (5sec), 72°C (15sec), and 30 cycles. The obtained PCR fragment was phosphorylated using Mighty Cloning Reagent Set (manufactured by Takara), self-ligated, and transformed into E. coli JM109 strain. A plasmid was extracted from the emerging colony and named pSK006. Hereinafter, the proB gene into which the three mutations described above have been introduced will be referred to as proB3m .

(1-2) Construction of an inducible expression plasmid To construct a ProB3m inducible expression plasmid, PCR amplification was performed using the pSK006 plasmid as a template and the primer set shown in SEQ ID NOs: 1 and 3 shown in Table 1. The reaction conditions were 98°C (10sec), 55°C (5sec), 72°C (15sec), and 30 cycles. PCR was performed using plasmid pBAD33 (provided by National Institute of Genetics, NBRP E. Coli strain) as a template and the primer set shown in SEQ ID NOs: 8 and 9 shown in Table 1. The reaction conditions were 98°C (10sec), 55°C (5sec), 72°C (30sec), and 30 cycles. The above two fragments were ligated and transformed into E. coli strain JM109. A plasmid was extracted from the emerging colony and named pSK007. The insertion direction was confirmed by colony PCR using the primer set shown in SEQ ID NOs: 3 and 10 shown in Table 1.

(1-3) Construction of plasmid for genome gene recombination Using the genomic DNA of E. coli strain W3110 (described above) as a template, PCR amplify the gene sequence containing proC with the primer set listed in SEQ ID NOs: 11 and 12 shown in Table 1. did. Similarly, the gene sequence containing putA was PCR amplified using the primer set described in SEQ ID NOs: 13 and 14. The reaction conditions for proC gene amplification are 98°C (10 sec), 55°C (5 sec), 72°C (10 sec), 30 cycles, and the reaction conditions for putA gene amplification are 98°C (10 sec), 55°C (5 sec), 72°C. (20 seconds) and 30 cycles. The two obtained PCR fragments were each phosphorylated using the Mighty Cloning Reagent Set (manufactured by Takara), and the pHAK1 plasmid (NITE P-02919) was transferred to the National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary (NPMD). ) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on March 18, 2019. E. coli transformed with the pHAK1 plasmid was cultured at 30°C unless otherwise specified. The ligation product was transformed into Escherichia coli JM109 strain, and plasmids were extracted from the colonies that appeared. The plasmid containing the proC gene was designated pSK008, and the plasmid containing the putA gene was designated pSK009.

Using pSK008 and pSK009 plasmids as templates, inverse PCR was performed to delete part of the gene sequence. The primer set shown in SEQ ID NOs: 15 and 16 in Table 1 was used for pSK008, and the primer set shown in SEQ ID NOs: 17 and 18 in Table 1 was used for pSK009, respectively. The reaction conditions were 98°C (10sec), 55°C (5sec), 72°C (40sec), and 30 cycles. The obtained PCR fragment was phosphorylated using Mighty Cloning Reagent Set (manufactured by Takara), self-ligated, and transformed into E. coli JM109 strain. Plasmids were extracted from the emerging colonies, and the plasmid containing the proC partial fragment was named pSK010, and the plasmid containing the putA partial fragment was named pSK011.

The ProBm2 expression cassette for insertion into the genome was obtained as follows. That is, PCR amplification was performed using pSK002 as a template and the primer set shown in SEQ ID NOs: 19 and 20 in Table 1. The obtained PCR fragment was phosphorylated using Mighty Cloning Reagent Set (manufactured by Takara). The ProBm2 expression cassette described above was ligated to the pSK008 or pSK009 DNA fragment that had been dephosphorylated without self-ligation. The ligation product was transformed into Escherichia coli JM109 strain, and plasmids were extracted from the colonies that appeared. The plasmid sequences were analyzed, and the plasmid with the ProBm2 expression cassette sandwiched between the proC fragment was named pSK012, and the plasmid with the ProBm2 expression cassette sandwiched between the putA fragment was named pSK013. Here, the gene sequence into which the ProBm2 expression cassette is inserted is designed to be partially deleted.

[Example 2] Construction of a genomic gene recombinant strain For the purpose of suppressing gene activity, genetic recombination was performed to delete part of the putA and proC genomic genes of E. coli using plasmids pSK010, pSK011, pSK012, and pSK013 for genomic gene recombination. was carried out. In addition, an operation was performed to insert a ProBm2 expression cassette into either of the two gene sequences.

(2-1) Creation of single-gene disrupted strain pSK010 or pSK011 was transferred to Escherichia coli AKC-016 strain (FERM P-22104) at the Patent Microorganism Depositary, National Institute of Technology and Evaluation (address: 2 Kazusa Kamatari, Kisarazu City, Chiba Prefecture). -5-8 Room 122) on April 20, 2011. International Deposit Number: FERM BP-11512) and cultured at 30°C on a plate medium containing kanamycin to obtain colonies. The obtained colony was grown into a liquid medium and cultured at 30°C for 3 hours. This culture solution was applied to a plate medium containing kanamycin and cultured at 44°C to obtain a plurality of colonies. The obtained colony was grown into a liquid medium and cultured at 30°C for 3 hours. This culture solution was applied to a plate medium containing 10% sucrose and cultured at 30°C to obtain a plurality of colonies. The genome proC sequence of the strain finally obtained by transformation with the pSK010 plasmid was analyzed, and AKSK01 was obtained as a strain in which the desired gene disruption had occurred. The genome putA sequence of the strain finally obtained by transformation with the pSK011 plasmid was analyzed, and the AKSK02 strain was obtained as a strain in which the desired gene disruption had occurred.

(2-2) Preparation of a two-gene disrupted strain To create a two-gene disrupted strain, the AKSK02 strain was transformed with the pSK010 plasmid and cultured at 30°C on a plate medium containing kanamycin to obtain colonies. The obtained colony was grown into a liquid medium and cultured at 30°C for 3 hours. This culture solution was applied to a plate medium containing kanamycin and cultured at 44°C to obtain a plurality of colonies. The obtained colony was grown into a liquid medium and cultured at 30°C for 3 hours. This culture solution was applied to a plate medium containing 10% sucrose and cultured at 30°C to obtain a plurality of colonies. The proC and putA sequences of the obtained colonies were analyzed, and the strain in which the desired gene disruption had occurred in both was designated as strain AKSK03.

(2-3) Preparation of a strain with genome insertion of the ProBm2 expression cassette To prepare an E. coli strain in which the two genes were disrupted and the ProBm2 expression cassette was inserted, the AKSK02 strain was transformed with the pSK012 plasmid, and the AKSK01 strain was transformed with the pSK013 plasmid. The cells were transformed and cultured at 30°C on a plate medium containing kanamycin to obtain colonies. The obtained colony was grown into a liquid medium and cultured at 30°C for 3 hours. This culture solution was applied to a plate medium containing kanamycin and cultured at 44°C to obtain a plurality of colonies. The obtained colony was grown into a liquid medium and cultured at 30°C for 3 hours. This culture solution was applied to a plate medium containing 10% sucrose and cultured at 30°C to obtain a plurality of colonies. Among the colonies obtained by transformation with pSK012, the genome proC was analyzed and the strain in which the desired gene insertion occurred was designated as the AKSK04 strain. Among the colonies obtained by transformation with pSK013, the genome putA gene sequence was analyzed, and the strain in which the desired gene insertion occurred was designated as the AKSK05 strain. Note that the AKSK04 strain has a proBm2 expression cassette inserted in the proC sequence, and the AKSK05 strain has a proBm2 expression cassette inserted in the putA sequence. The gene sequence into which the proBm2 expression cassette is inserted is designed to be partially deleted, so in the AKSK04 strain, the proC gene is disrupted in addition to the putA gene, and in the AKSK05 strain, the proC gene is also disrupted. The putA gene is also disrupted.

[Example 3] Cultivation and analysis of produced strains (Example 3-1)
The AKSK03 strain or the AKC-016 strain was transformed with the pSK004 plasmid (these will be referred to as the AKSK06 strain and the AKSK07 strain, respectively). For comparison, non-transformed AKC-016 and AKSK03 strains were also prepared and cultured overnight at 37°C on an LB plate medium. The obtained colony was grown into 2 mL of LB medium and cultured with shaking at 37° C. overnight. 50 uL of this culture was added to 5 mL of M9 medium placed in a 15 mL round bottom tube. The turbidity (OD600) was observed over time, and when it exceeded 0.4, L-arabinose was added and cultured with shaking at 37°C for an additional 20 hours. The supernatant obtained by centrifuging the collected culture solution was diluted 4 times with 1% formic acid. This diluted solution was subjected to MF filtration using a Chromato Disk (manufactured by Shimadzu DLC) and analyzed by LCMS. The analysis conditions are shown in Table 3.

A total ion chromatography chart of the culture supernatant is shown in FIG. In the culture samples of AKC-016 strain, AKSK07 strain, and AKSK03 strain, no peak indicating glutamic acid semialdehyde ("GSA" in the figure) or proline-5-carboxylic acid ("P5C" in the figure) could be detected. . On the other hand, in the culture sample of strain AKSK06, peaks indicating glutamic acid semialdehyde (GSA) and proline-5-carboxylic acid (P5C) were detected.

The concentrations of glutamate semialdehyde and proline-5-carboxylic acid excreted into the culture supernatant were determined from the literature (Ruiter et. al., Plant Physiol., American Society of Plant Biologists, 1983, 73, 525-528.) Quantitated according to the method described. The extinction coefficient of the reactant with o-aminobenzaldehyde was calculated as ε=2,300 M−1 cm−1. The results are shown in Table 4.

(Example 3-2)
Strains AKSK04 and AKSK05 were grown into 2 mL of LB medium and cultured with shaking at 37°C overnight. 50 uL of this culture was added to 5 mL of M9 medium in a 15 mL round bottom tube, and cultured with shaking at 37° C. for 22 hours. AKC-016 strain was used as a control and cultured under the same conditions. The supernatant obtained by centrifuging the collected culture solution was diluted 4 times with 1% formic acid. This diluted solution was subjected to MF filtration using a chromato disk (manufactured by Shimadzu GLC), and analyzed by LCMS.

A total ion chromatography chart of the AKC-016 strain, AKSK04 strain, and AKSK05 strain culture supernatant is shown in FIG. In the charts of AKSK04 and AKSK05, a peak of L-pyroglutamic acid was detected. In strain AKSK04, peaks of glutamic acid semialdehyde and pyrroline-5-carboxylic acid were further detected.

The concentrations of glutamic acid semialdehyde and proline-5-carboxylic acid discharged into the culture supernatant were quantified. The results are shown in Table 5.

INDUSTRIAL APPLICATION This invention can be utilized for the industrial fermentation production of glutamic acid-5-semialdehyde, pyrroline-5-carboxylic acid, and/or L-pyroglutamic acid.

Claims (13)

Modifications are made so that the expression of the gene encoding pyrroline-5-carboxylic acid dehydrogenase and the gene encoding pyrroline-5-carboxylic acid reductase is suppressed ,
Further modifications are made to enhance the activity of glutamate-5-phosphate synthase,
Further modification such that the activity of glutamate-5-phosphate synthase is enhanced increases the expression level of glutamate-5-phosphate synthase,
A recombinant microorganism that is Escherichia coli. The recombinant microorganism according to claim 1, wherein the further modification releases feedback inhibition of glutamate-5-phosphate synthase. The gene encoding the pyrroline-5-carboxylic acid dehydrogenase is
・DNA consisting of the base sequence shown in SEQ ID NO: 27,
・DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 27, and encodes a protein that has pyrroline-5-carboxylic acid dehydrogenase activity,
・DNA consisting of a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 27 and encoding a protein having pyrroline-5-carboxylic acid dehydrogenase activity,
- DNA encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted, and/or added to the amino acid sequence of the protein encoded by the base sequence shown in SEQ ID NO: 27. DNA encoding a protein having pyrroline-5-carboxylic acid dehydrogenase activity, or DNA consisting of a degenerate isomer of the base sequence shown in SEQ ID NO: 27.
and
and/or
The gene encoding the pyrroline-5-carboxylic acid reductase is
・DNA consisting of the base sequence shown in SEQ ID NO: 28,
- A DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 28, and encodes a protein having pyrroline-5-carboxylic acid reductase activity,
- A DNA consisting of a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 28, and encoding a protein having pyrroline-5-carboxylic acid reductase activity,
- DNA that encodes a protein consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted, and/or added to the amino acid sequence of the protein encoded by the base sequence shown in SEQ ID NO: 28. DNA encoding a protein having pyrroline-5-carboxylic acid reductase activity, or DNA consisting of a degenerate isomer of the base sequence shown in SEQ ID NO: 28.
The recombinant microorganism according to claim 1 or 2, which is. said pyrroline-5-carboxylic acid dehydrogenase is selected from the enzymes expressed by EC 1.2.1.88, and/or said pyrroline-5-carboxylic acid reductase is selected from the enzymes expressed by EC 1.5.1.2. The recombinant microorganism according to any one of claims 1 to 3, wherein the recombinant microorganism is selected from the following enzymes. The pyrroline-5-carboxylic acid dehydrogenase has an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and has pyrroline-5-carboxylic acid dehydrogenase activity. is a protein and/or
The pyrroline-5-carboxylate reductase is a protein having an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 24 and having pyrroline-5-carboxylate reductase activity. The recombinant microorganism according to any one of claims 1 to 4. The glutamate-5-phosphate synthase is
・DNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 29 and encoding a protein having glutamic acid-5-phosphate synthase activity, or
- DNA that encodes a protein consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted, and/or added to the amino acid sequence of the protein encoded by the base sequence shown in SEQ ID NO: 29. DNA encoding a protein having glutamate-5-phosphate synthase activity
The recombinant microorganism according to any one of claims 1 to 5, encoded by. Any one of claims 1 to 6, which produces a protein having an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 25 and having glutamate-5-phosphate synthase activity. The recombinant microorganism according to item 1. For the amino acid sequence shown in SEQ ID NO: 25,
Substitution of the 107th aspartic acid residue with an asparagine residue,
has an amino acid sequence that has 90% or more sequence identity to an amino acid sequence that includes the substitution of an alanine residue at position 117 with a valine residue and the substitution of a glutamic acid residue at position 143 with a lysine residue, The recombinant microorganism according to any one of claims 1 to 7, which produces a protein having glutamate-5-phosphate synthase activity. For the amino acid sequence shown in SEQ ID NO: 25,
Substitution of the 107th aspartic acid residue with an asparagine residue,
has one or more mutations selected from the group consisting of substitution of the 117th alanine residue with a valine residue, and substitution of the 143rd glutamic acid residue with a lysine residue, and glutamic acid-5-phosphorus The recombinant microorganism according to any one of claims 1 to 8, which produces a protein having acid synthase activity. The recombinant microorganism according to any one of claims 1 to 9, wherein the gene encoding glutamate-5-phosphate synthase is contained as an operon with the gene encoding glutamate-5-semialdehyde reductase. . A method for producing glutamic acid-5-semialdehyde, which comprises culturing the recombinant microorganism according to any one of claims 1 to 10. A method for producing pyrroline-5-carboxylic acid, which comprises culturing the recombinant microorganism according to any one of claims 1 to 10. A method for producing L-pyroglutamic acid, which comprises culturing the recombinant microorganism according to any one of claims 1 to 10.

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